Synthesis of heterogeneous mRNA-like RNA and low-molecular-weight RNA before the midblastula transition in embryos of Xenopus laevis

It has been proposed and is now widely accepted that in Xenopus laevis embryogenesis RNA synthesis starts only at and after 12 rounds of cleavage, at the time of the midblastula transition (MBT). In this report, however, we provide evidence that RNA synthesis takes place prior to the MBT stage in normally developing Xenopus embryos. In the present experiments, we cultured fertilized eggs in 80 mM phosphate buffer and loosened the adhesion between blastomeres, so that [3H]uridine could be incorporated into blastomeres from the surrounding medium. By this method and also by microinjection of [3H]GTP, we found that embryos synthesize heterogeneous, nonribosomal, high-molecular-weight RNAs and a relatively small amount of low-molecular-weight RNA as early as the sixth cleavage. RNAs synthesized were not of mitochondrial origin, and the synthesis was sensitive to actinomycin D and alpha-amanitin. From these results we conclude that mRNA-like RNA and low-molecular-weight RNA start to be synthesized during the cleavage stage.     

Expression of cell adhesion molecule E-cadherin in Xenopus embryos begins at gastrulation and predominates in the ectoderm

The expression of the Ca2+-dependent epithelial cell adhesion molecule E-cadherin (also known as uvomorulin and L-CAM) in the early stages of embryonic development of Xenopus laevis was examined. E-Cadherin was identified in the Xenopus A6 epithelial cell line by antibody cross-reactivity and several biochemical characteristics. Four independent mAbs were generated against purified Xenopus E-cadherin. All four mAbs recognized the same polypeptides in A6 cells, adult epithelial tissues, and embryos. These mAbs inhibited the formation of cell contacts between A6 cells and stained the basolateral plasma membranes of A6 cells, hepatocytes, and alveolar epithelial cells. The time of E-cadherin expression in early Xenopus embryos was determined by immunoblotting. Unlike its expression in early mouse embryos, E-cadherin was not present in the eggs or early blastula of Xenopus laevis. These findings indicate that a different Ca2+-dependent cell adhesion molecule, perhaps another member of the cadherin gene family, is responsible for the Ca2+-dependent adhesion between cleavage stage Xenopus blastomeres. Detectable accumulation of E-cadherin started just before gastrulation at stage 9 1/2 and increased rapidly up to the end of gastrulation at stage 15. In stage 15 embryos, specific immunofluorescence staining of E-cadherin was discernible only in ectoderm, but not in mesoderm and endoderm. The ectoderm at this stage consists of two cell layers. The outer cell layer of ectoderm was stained intensely, and staining was localized to the basolateral plasma membrane of these cells. Lower levels of staining were observed in the inner cell layer of ectoderm. The coincidence of E-cadherin expression with the process of gastrulation and its restriction to the ectoderm indicate that it may play a role in the morphogenetic movements of gastrulation and resulting segregation of embryonic germ layers.     

Expression of a novel cadherin (EP-cadherin) in unfertilized eggs and early Xenopus embryos

Two distinct cadherin cDNA clones of Xenopus laevis were isolated from a stage 17 embryo cDNA library. Analysis of the complete deduced amino acid sequences indicated that one of these molecules is closely homologous to chicken and mouse N-cadherin, while the other displays comparable homology to both E- and P-cadherins and was thus denoted EP-cadherin. This molecule has an apparent relative molecular mass of 125 x 10(3) (compared to approx. 138 x 10(3) or approx. 140 x 10(3) of E-cadherin and N-cadherins, respectively). Northern and Western blot analyses indicated that N-cadherin is first expressed at the neurula stage while EP-cadherin is the only cadherin detected in unfertilized eggs and cleavage stage embryos. Immunolabeling of Xenopus eggs with antibodies prepared against a fusion protein, containing a segment of EP-cadherin, indicated that the protein is highly enriched at the periphery of the animal hemisphere. EP-cadherin was also found in A6 epithelial cells derived from Xenopus kidneys, and was apparently localized in the intercellular adherens junctions.     

Presence of a nucleus or nucleus-deriving factors is indispensable for the formation of the spindle, the diastema and the cleavage furrow in the blastomere of the Xenopus embryo

The present study examines the indispensability of a nucleus or nucleus-deriving factors in the induction of cleavage in Xenopus eggs by testing cleavage in Xenopus eggs fertilized with ultraviolet (UV)-damaged sperm and deprived of the female nucleus. These eggs, which contain only one UV-damaged nucleus with one set of centrioles, undergo unique cleavages. Cleavage takes place in only one of the two blastomeres formed by the immediately preceding cleavage. Histologically, only one nucleus, which does not appear to be organized into typical chromosomes, is found in one of the two blastomeres formed by the immediately preceding cleavage. The typical bipolar spindle and the diastema, or a slit of astral rays, are formed in the blastomere that contains the nucleus. By contrast, only asters lacking the spindle and the diastema are formed in the remaining blastomeres, which do not contain a nucleus. The same results are obtained in eggs that contain two UV-damaged nuclei with one set of centrioles. In these eggs, cleavage appears to occur in one or two blastomeres that contain either or both of the nuclei and one bipolar spindle. In eggs that contain one intact and one UV-damaged nuclei, cleavage takes place quite normally with each blastomere containing one nucleus or one set of chromosomes as well as one bipolar spindle. Thus, there is a very close correlation between the presence of a nucleus and the formation of the mitotic spindle, the diastema and the cleavage furrow in the blastomeres of Xenopus embryos. We conclude that the presence of a nucleus or nucleus-deriving factors is indispensable for the formation of the bipolar spindle, the diastema and the cleavage furrow in the blastomeres of the Xenopus embryos.     

Fates of Animal-Dorsal Blastomeres of Eight-Cell Stage Xenopus Embryos Vary according to the Specific Patterns of the Third Cleavage Plane

The third cleavage plane in typical Xenopus embryos is horizontal. However, there are numbers of cases in which the third cleavage plane slants and yet the embryo develops normally. Pairs of animal-dorsal (AD) blastomeres of eight-cell stage Xenopus embryos with horizontal or oblique third cleavage plane were marked by intracellular injection of fluorescein dextran amine in order to locate their progeny. In neurulae, progeny of AD blastomeres was found mainly along the dorsal midline forming longitudinal clonal bands along the midline in the neural plate and the mesoderm underneath. AD blastomeres with oblique third cleavage plane further yielded the ventral endo-mesoderm in the head. On the other hand, they formed narrower clonal bands in the anterior ectoderm compared with AD blastomeres with horizontal third cleavage plane. Thus the fates of animal-dorsal brastomeres of eight-cell stage Xenopus embryos vary according to the specific patterns of the third cleavage plane. This indicates that the third cleavage in the Xenopus embryo does not affect the normal fate of each region of the embryo presumed at the eight-cell stage.     

Development of reconstituted mouse embryos produced from the cytoplast of bisected oocytes or pronuclear-stage embryos and single blastomeres of 2-cell stage embryos

The present study investigated the in vitro developmental potential of reconstituted mouse embryos produced from the cytoplast of pronuclear-stage embryos or oocytes and single blastomeres of 2-cell stage embryos by electrofusion. The cytoplast of pronuclear-stage embryos and oocytes were obtained by manual bisection with a fine glass needle under a dissecting microscope. The fusion rates of the reconstituted embryos produced from the cytoplast of oocytes and single blastomeres of 2-cell stage embryos (O-SB2: 38.1 and 41.5%) were significantly lower than those produced from the cytoplast of pronuclear-stage embryos and single blastomeres of 2-cell stage embryos (P-SB2: 91.2 and 97.6%; P<0.001). Reconstituted embryos were encapsulated in alginate gel and were cultured for 96 hours. Similarly, the cleavage and development rates to the blastocyst stage of O-SB2 (56.3, 61.2 and 2.0, 3.1%, respectively) were significantly lower than those of the P-SB2 (91.0, 91.2 and 18.6, 20.7%; respectively, P<0.05). The cleavage and development rates to the blastocyst stage (61.2 and 2.0%) of reconstituted embryos produced from single blastomeres of late 2-cell stage embryos and oocyte cytoplast improved after activation by ethanol treatment (76.1 and 21.7%). However, the use of single blastomeres of early 2-cell stage embryos as nuclear donors did not enhance the cleavage and development rates of the reconstituted embryos to the blastocyst stage.     

Blastomeres show differential fate changes in 8-cell Xenopus laevis embryos that are rotated 90° before first cleavage

To study the mechanisms of dorsal axis specification, the alteration in dorsal cell fate of cleavage stage blastomeres in axis-respecified Xenopus laevis embryos was investigated. Fertilized eggs were rotated 90° with the sperm entry point up or down with respect to the gravitational field. At the 8-cell stage, blastomeres were injected with the lineage tracers, Texas Red- or FITC-Dextran Amines. The distribution of the labeled progeny was mapped at the tail-bud stages (stages 35–38) and compared with the fate map of an 8-cell embryo raised in a normal orientation. As in the normal embryos, each blastomere in the rotated embryos has a characteristic and predictable cell fate. After 90° rotation the blastomeres in the 8-cell stage embryo roughly switched their position by 90°, but the fate of the blastomeres did not simply show a 90° switch appropriate for their new location. Four types of fate change were observed: (i) the normal fate of the blastomere is conserved with little change; (ii) the normal fate is completely changed and a new fate is adopted according to the blastomere's new position; (iii) the normal fate is completely changed, but the new fate is not appropriate for its new position; and (4) the blastomere partially changed its fate and the new fate is a combination of its original fate and a fate appropriate to its new location. According to the changed fates, the blastomeres that adopt dorsal fates were identified in rotated embryos. This identification of dorsal blastomeres provides basic important information for further study of dorsal signaling in Xenopus embryos.     

Polar asymmetry in the organization of the cortical cytokeratin system of Xenopus laevis oocytes and embryos

We have used whole-mount immunofluorescence microscopy of late-stage Xenopus laevis oocytes and early embryos to examine the organization of their cortical cytokeratin systems. In both mature oocytes and early embryos, there is a distinct animal-vegetal polarity in cytokeratin organization. In mature (stage-VI) oocytes, the cytokeratin filaments of the vegetal region form a unique, almost geodesic network; in the animal region, cytokeratin organization appears much more variable and irregular. In unfertilized, postgerminal vesicle breakdown eggs, the cortical cytokeratin system is disorganized throughout both animal and vegetal hemispheres. After fertilization, cytokeratin organization reappears first in a punctate pattern that is transformed into an array of oriented filaments. These cytokeratin filaments appear first in the vegetal hemisphere and are initially thin. Subsequently, they form bundles that grow thicker through the period of first to second cleavage, at which point large cytokeratin filament bundles form a loose, fishnet-like system that encompasses the vegetal portion of each blastomere. In the animal region, cytokeratin filaments do not appear to form large fibre networks, but rather appear to be organized into a system of fine filaments. The animal-vegetal polarity in cytokeratin organization persists until early blastula (stage 5); in later-stage embryos, both animal and vegetal blastomeres possess qualitatively similar cytokeratin filament systems. The entire process of cytokeratin reorganization in the egg is initiated by prick activation. These observations indicate that the cortical cytoskeleton of Xenopus oocytes and early embryos is both dynamic and asymmetric.     

Differential distribution of ganglioside GM1 and sulfatide during the development of Xenopus embryos

A frozen section technique for frog oocytes was developed without using any organic solvent. It was applied to examine the distribution of acidic glycosphingolipids (ganglioside GM1 and sulfatide) in Xenopus oocytes, eggs and embryos by indirect immunofluorescence microscopy with specific monoclonal antibodies against the acidic glycolipids. Although glycolipids are generally present on the cell surface, GM1 and sulfatide were distributed in the cytoplasm of animal and vegetal hemispheres, respectively, of the fully grown oocytes and oviposited and fertilized eggs. In blastula, GM1 was present on the cell boundaries and in the Golgi of the blastomeres of animal hemisphere and marginal zone, whereas the staining of the outermost layer of animal blastomeres became faint or negligible at stage 9. Sulfatide in blastula was still observed in vegetal blastomeres. In gastrula, GM1 was distributed in the inner layer of ectoderm and the involuting mesoderm. In neurula, GM1 was concentrated in the dorsal midline including the closing neural tube, notochord and somites, while sulfatide was present in endoderm. The unique distribution of GM1 and sulfatide in oocytes, eggs and early embryos may help to elucidate one aspect of the biochemical bases laid on the animal–vegetal polarity.     

Talin and vinculin in the oocytes, eggs, and early embryos of Xenopus laevis: a developmentally regulated change in distribution

We have investigated the expression and distribution of talin and vinculin in the oocytes, eggs, and embryos of Xenopus laevis. Antibodies to the previously characterized avian proteins stain several different Xenopus cell types identically by immunofluorescence: adhesion plaques of cultured kidney (A6) cells, the cell peripheries of oviduct cells, and the postsynaptic neuromuscular junctions of tadpole tail muscle fibers. These antibodies also identify cognate proteins of the appropriate sizes on immunoblots of A6 cell and oviduct lysates. Using these antibodies on ovarian tissue, we find talin to be highly localized at the cortices of oocytes and vinculin to be in the oocyte cytoplasm and absent from the oocyte cortex. In the cells of the ovarian layers that surround the oocytes, talin and vinculin can be detected as soluble and cytoskeletal components. Vinculin is first detectable as a cytoskeletal component in eggs, appearing some time during or between oocyte maturation and oviposition. During early embryo development, talin and vinculin are colocalized in the cortex of cleavage furrows and blastomeres. Thus, Xenopus oocytes and eggs display different distributions of talin and vinculin. The change from unlinked localization to colocalization appears to be developmentally regulated, occurring during the transition from oocyte to egg.     

Asters play only a dispensable role in the induction of the cleavage furrow in the blastomeres of early Xenopus embryos

Kuroda et al. (2001) of our laboratory have previously revealed that exposure of early Xenopus embryos to 150 mm urethane results in complete suppression of formation of the asters and the cleavage furrow, as well as significant reduction of the size of the spindle in the blastomeres, allowing only 1 or 2 cycles of mitosis but not cytokinesis. In the course of closer examination of the effect of urethane on the cleavage of blastomeres of early Xenopus embryos, we unexpectedly discovered that exposure of early Xenopus embryos to 75 mm urethane did not prevent cell division at all, though asters were not detected in the blastomeres. Instead, they contained a spindle that appeared rather normal. They also formed the diastema, a thin yolk-free structure, which is considered to play an essential role in the induction of the cleavage furrow. Essentially the same results were obtained in the exposure of embryos to vinblastine, a well-known microtubule inhibitor: exposure of embryos to 20 micro g/mL vinblastine resulted in complete suppression of cleavage of the blastomeres, where formation of both the spindle and asters were perfectly suppressed. By contrast, exposure of embryos to 5 microg/mL vinblastine did not prevent cleavage in the blastomeres though asters were not detected, whereas the rather normal spindle was formed. Thus, there was a close correlation between the formation of the normal spindle, not asters, and that of the cell division furrow and the diastema in the blastomeres of early Xenopus embryos. We suggest that while the spindle plays an essential role, asters are likely to play only a dispensable role in the induction of the cleavage furrow in even very large cells like the blastomeres of early Xenopus embryos.     

Expression of DNA ligases I and II during oogenesis and early development of Xenopus laevis.

We have analyzed the expression of DNA ligase I protein during oogenesis and early development of Xenopus laevis. The protein is already present in stage I oocytes and then accumulates throughout oogenesis to reach a steady state level by stage VI. It remains at this level at least until tadpole stage. In stage VI oocytes DNA ligase I protein is almost exclusively localized in the germinal vesicle. We have partially purified a DNA ligase II activity from stage VI oocytes, unfertilized eggs, and stage 8 embryos. An 80-kDa polypeptide can be specifically adenylated in all three purified extracts. It is not recognized by antibodies directed against DNA ligase I and is active on oligo(dT)-poly(rA) substrate. It could therefore represent DNA ligase II protein. The presence of both DNA ligases I and II in oocytes and embryos is inconsistent with the DNA ligase model that had been previously proposed for amphibia.     

Cell proliferation in the ectoderm of the Xenopus embryo: development of substratum requirements for cytokinesis

The requirements for cell division in ectodermal blastomeres of the early Xenopus embryo were studied. Isolated blastomeres divide autonomously on nonadhesive agar in a simple salt solution up to the midblastula stage. After the midblastula transition, cell-cell contact is required for blastomere division. In isolated blastomeres of that stage, cytokinesis fails, but nuclear division continues normally for some time. Cell-cell contact as a prerequisite for blastomere division can be replaced by culturing blastomeres on an appropriate substratum. Clonal growth of isolated blastomeres is supported by a variety of protein substrata, indicating rather unspecific substratum requirements. Different substrata which do not support blastomere division can affect different steps in cytokinesis.     

Occurrence of pre-MBT synthesis of caspase-8 mRNA and activation of caspase-8 prior to execution of SAMDC (S-adenosylmethionine decarboxylase)-induced, but not p53-induced, apoptosis in Xenopus late blastulae

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus fertilized eggs activates caspase-9 and executes maternal program of apoptosis shortly after midblastula transition (MBT). We find that overexpression of caspase-8 and p53, like that of SAMDC, induces apoptosis in Xenopus late blastulae. The apoptosis induced by p53 was abolished by injection of mRNA for xdm-2, a negative regulator of p53, and by injection of a peptide inhibitor or a dominant-negative type mutant of caspase-9, but not caspase-8. The apoptosis induced by SAMDC was not abolished by injection of xdm-2 mRNA, but was abolished by injection of a peptide inhibitor or a dominant-negative type mutant mRNA of both caspase-9 and caspase-8. Unlike caspase-9 mRNA, caspase-8 mRNA did not occur as a maternal mRNA rather induced to be expressed during cleavage stage (pre-MBT stage) by overexpression of SAMDC but not p53. Furthermore, while activities to process procaspase-8 and procaspase-9 appeared in SAMDC-overexpressed apoptotic embryos, the activity to process procaspase-8 did not appear in p53-overexpressed apoptotic embryos. We conclude there are at least two pathways in the execution of the maternal program of apoptosis in Xenopus embryos; one being through do novo expression of caspase-8 gene during cleavage stage, and the other without involvement of caspase-8.     

Genesis of Epidermal Action Potentials in Cytokinesis Arrested Xenopus Embryos

When Xenopus embryos were treated continuously with cytochalasin B (3–10 μg/ml) from the 8 cell stage, cleavage arrested embryos in various degrees were observed. In 3–5 μg/ml cytochalasin B, cytokinesis was inhibited at the midblastula stage and pigment granules remained at the cell cortex of the animal pole. These cells showed epidermal like action potentials when the control embryos (St. 26/28) generated epidermal action potentials. In 5–7 μg/ml cytochalasin B, furrows, following their formation at early cleavage stages, regressed and no further cleavage from the 16 cell stage to morula stage took place. The pigment granules were dispersed throughout the interior of the cytoplasm. These cells showed no epidermal action potentials. Thus, it is considered that cytokinesis per sé , following the midblastula stage, is not a prerequisite for the genesis of epidermal action potentials, and that chronological times corresponding to the tailbud larva stage and a stable structure of the cellular cortex are required to bring about these potentials.     

Germinal vesicle components are not required for the cell-cycle oscillator of the early starfish embryo

We show that certain events of the cell cycle can still occur in starfish oocytes or fertilized eggs from which the germinal vesicle (the prominent nucleus of prophase-arrested oocytes) has been removed before the induction of meiotic maturation. Two meiotic asters develop following hormonal induction of meiotic maturation in these enucleated oocytes. The asters then divide to form a transient tetrapolar figure. When enucleated oocytes are fertilized, the sperm centrosome duplicates at the times corresponding to each cleavage in control nucleated embryos. Periodic changes in the organization of the asters and in the morphology of the cell surface also occur in synchrony with controls. Decondensation of the sperm nucleus, spindle formation, and cleavage do not occur when enucleated oocytes are fertilized. Ultimately the number of asters increases to approximately 520 (about 2(9] before the pseudo-embryo arrests and cytolyzes. Fertilized eggs from which both pronuclei but not the sperm aster have been removed undergo nine cleavages and then cease cell division. The cessation of division may be related to the events that cause the midblastula transition after seven cleavages in normal nucleated embryos.